The RAD6 mediated post-replication and repair and mutagenesis pathway is critical for genome stability in both yeast and mammalian cells. The RAD6 pathway is composed of several subbranches that interface with other repair pathways to partition damage between error-free and mutagenic outcomes. Without this pathway cells are immutable. Despite the obvious importance of mutation to human health, our understanding of this pathway is currently limited. We are developing systematic approaches to identify genes of this pathway and have recently reported several new genes of the error-free subbranch. However, in order to understand mutation it is critical to understand both the error-free and mutagenic subbranches, as well as how damage is partitioned between them and other pathways. We therefore propose the further characterization of three, already identified genes, which function in the error-free response, or at the interface with recombination or the cell cycle. We also propose the extension of the approach to identify and characterize important genes of the mutagenic subbranch. The specific aims of the proposed research are as follows: 1) Characterize the roles played by DOA1, ESC4, and WSS1 in the error-free response. 2) Generate a set of candidate mutagenesis genes using genome wide screens. 3) Determine authentic mutagenesis genes and identify the most critical for mutation. 4) Characterize the roles played by the mutagenesis genes in the mutagenic response. These studies are expected to help understand when and how eukaryotic cells mutate, and will contribute to our understanding of issues that are of fundamental significance to human health, such as aging and cancer.